NAD, and NADH are the most popular coenzymes included in the body, and they participate in oxidation-reduction reactions of various dehydrogenases by means of reversible changes between AND and NADH which receive or give a hydrogen atom.
NAD and NADH can be detected or analyzed with a potent sensitivity in the aid of signal amplification system utilizing the enzymatic cycling between NAD and NADH. This signal amplification system is also applied extensively to phosphatase detection or analysis system in the fields of immunoassay and tissue staining. In spite of that the method of detection or analysis of NAD or NADH associated with the signal amplification system is colorimetric analysis, it is now an excellent method in the field of immunoassay, especially, in the measurement of alkaline phosphatase, since it can provide a potent detection sensitivity which is comparable to that in fluorescence technique (or fluorescent antibody technique) or color development technique [edited by Isikawa et al., Kohso Men-eki Sokuteihoh (in Japanese), the third edition, pp. 58-60, published by Igakushoin].
In the method, nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADP) or its reduced form (hereinafter abbreviated as NADPH) is coverted into NAD or NADH by catalytic action of phosphatase, the resultant NAD or NADH is detected by means of signal amplification system thereby presence of phosphatase if detected in a potent sensitivity.
As will be seen from the reaction diagram shown hereunder, when NADP is used as a substrate for phosphatase, for example, NAD is produced by catalytic action of phosphatase, the resultant NAD act as a coenzyme for oxidation reaction from ethanol to acetoaldehyde, in the presence of alcohol dehydrogenase (hereinafter abbreviated as ADH) and its substrate (for example, ethanol which is hereinafter abbreviated as EtOH), at this occasion NAD is reduced to NADH. The resultant NADH is oxidized to produce NAD in the presence of tetrazolium dye and its reductase (diaphorase), and at the same time terazolium dye is reduced to produce formazan as the color-development signal. Under the conditions mentioned above, the oxidation-reduction reaction between NAD and NADH is repeated to thereby gradually increase the signal intensity. (C. H. Self et al., Clinica Chimica Acta, Vol 148, pp. 119-124, 1985). ##STR1##
In the reaction system described, the enzyme participating in the reduction reaction of NAD to NADH must be the one which recognize NAD but does not recognize NADP, i.e. which is activated only by NAD but is not activated by NADP. Usually ADH is used in view of its potent specificity to NAD.
As will be seen from the reaction diagram shown hereunder, when NADPH is used as a substrate for phosphatase, a color development signal is generated and amplified in the same manner (principle) as described with respect to NADP in the above. In this case, it is necessary to use an enzyme participating in oxidation reaction of NADH to NAD must be the one which solely recognize NADH but do not recognize NADPH, and usually NADH-depending (or NADH-specific) diaphorase is used (F. J. Dhahir et al., Clinical Chemistry, Vol. 38 (2), pp. 227-232, 1992). ##STR2##
In measurement of phosphatase utilizing signal amplification system of NAD or NADH, a first reagent participating in catalytic reaction of phosphatase and a second reagent participating in signal amplification system are utilized. That is, the first reagent includes NADP or NADPH, which is the substrate for phosphatase, as the main component, and the second reagent includes enzymes for the enzymatic cycling of NAD or NADH and for enhancement of the signal amplification (for example, ADH and diaphorase) and their substrates (for example, alcohol and tetrazolium) as the main components.
The second reagent which participate in signal amplification, however, tends to show non-specific color development (hereinafter, this phenomenon is occasionally called a reagent blank) of tetrazolium dye, when all the components of the second reagent are preserved in a mixture. For this reason, it is common that the second reagent is further separated into two parts or compositions in preservation, and this necessitates to mixing them immediately before actual use.